Choosing the right Cas9 nuclease

With so many to choose from - how do you know which Cas9 is best for your experiment?

The Streptoccocus pyogenes CRISPR-Cas9 system requires guide RNA and nuclease components to create a double-strand cut, and subsequent edit, to genomic DNA. Guide RNA may consist of a dual RNA system of gene-specific CRISPR RNA (crRNA) with trans-activating crRNA (tracrRNA), or a gene-specific single guide RNA (sgRNA) that combines the crRNA and tracrRNA in a single transcript. The Cas9 nuclease component is available in multiple formats to allow for experimental flexibility and maximal success for particular cell types and applications. Since optimal activity of the RNA-guided Cas9 endonuclease is critical for successful gene editing, below is a list of considerations that can help direct you to the type of Cas9 nuclease best suited for your editing needs:

Vector-based Cas9: plasmid or lentiviral particles

Vector-based Cas9 expression systems are useful for researchers who would like to enrich for Cas9-expressing cells or wish to establish a stable cell line. The Dharmacon Edit-R™ selection of vector-based Cas9 nuclease comes in two general formats:

Cas9 expression plasmid

With a multitude of choices for promoters and reporter, the Edit-R Cas9 Nuclease Expression plasmid option is good for customers with easily transfectable cells who would like the ability to enrich their cell population by antibiotic selection or fluorescent reporter cell sorting following a transient co-transfection of Cas9 plasmid with synthetic crRNA:tracrRNA.

Lentiviral Cas9 Nuclease

Edit-R Lentiviral Cas9 Nuclease is a good option for gene editing in difficult-to-transfect cells due to the wide dynamic range of lentiviral transduction. This option can also be used to establish a Cas9-expressing stable cell line for arrayed crRNA:tracrRNA or pooled sgRNA screening applications.

Vector-based Cas9: promoter options

The activity of a particular promoter can result in variable Cas9 expression levels and potentially low editing efficiency. Vector-based Edit-R Cas9 Nuclease products are offered with a choice of six well-characterized constitutive cellular promoters so you can choose an optimal promoter for your cells. The Edit-R Lentiviral Cas9 Nuclease is also available with an inducible promoter to support creation of stable cell lines with minimal background expression, or for temporal control over Cas9 expression for wide-ranging experimental applications.

DNA-free Cas9: mRNA or protein

DNA-free Cas9 is most commonly used with synthetic crRNA:tracrRNA and chosen by researchers that would like to avoid unwanted vector DNA integration into their genomic DNA. Cas9 mRNA or protein is transiently available for editing events which means that these nuclease sources do not have the off-targeting potential of constitutive Cas9 expression. For that reason, CRISPR-Cas9 utilizing mRNA or protein are ideal for applications such as knockin of a fluorescent reporter using HDR or knockout cell line generation. Both are amenable to lipid-based transfection reagents or electroporation to support a wide variety of cell types. Finally, mRNA and protein are suited for experiments where available Cas9 plasmid or lentiviral particle promoters are not compatible in their cell line or model organism.

Cas9 protein

The main advantage of delivering Edit-R Cas9 protein with crRNA:tracrRNA is that the translated protein is immediately available for complexing with the guide RNA and consequent editing in the cell. This is often a factor in applications where the cells lack robust translational machinery such as stem cells or embryonic model systems.

Cas9 mRNA

Purified, translation-ready Edit-R Cas9 Nuclease mRNA, in combination with crRNA:tracrRNA is an economical option for researchers who want to perform DNA-free editing for generation of knockout or knockin clonal cell lines, or arrayed screening.